Toll-like receptors (TLRs) are an evolutionarily conserved family of pattern recognition receptors that initiate inflammatory responses essential for controlling infection and activating adaptive immune system. Ten human TLRs have been discovered and cloned each of which recognize specific viral, microbial or fungal products. TLR2 recognizes a large repertoire of ligands and it has been shown that TLR1 or TLR6 is required as a partner for TLR2 activity. The broad aim of this proposal is to discern the mechanism of microbial recognition of TLRs 1,2 and 6 that lead to inflammatory responses. The specific aims of this proposal are to: 1) Define the structural determinants of microbial products that are required for TLR2/1 and TLR2/6 recognition through the use of reconstitution and reporter assays as well as siRNA-mediated knock down studies in myelomonocytic cells. 2) Determine the region of the extracellular domain of TLR1 and TLR6 that is important for enabling TLR2 to discriminate between microbial agonists through domain swapping of the extracellular regions between these receptors. 3) Define the colocalization and examine the possible agonist-induced physical interactions between TLR2 with either TLR1 or TLR6. This latter aim is accomplished through fluorescence microscopy and FRET using CFP and YFP fusion proteins of these TLRs. The results of these studies have implications for the rational design of better vaccine adjuvants and in the control of fatal inflammatory conditions such as septic shock.